Increasing responses to checkpoint inhibitors by extracorporeal apheresis

ABSTRACT

The invention provides means, methods, and compositions of matter useful for enhancing tumor response to checkpoint inhibitors. In one embodiment, the invention teaches utilization of extracorporeal apheresis, specifically removal of various tumor derived, or tumor microenvironment derived immunological “blocking factors”. In one embodiment the invention provides the removal of soluble TNF-alpha receptors (sTNF-Rs) as a means of augmenting efficacy of immune checkpoint inhibitors. In one specific embodiment removal of sTNF-Rs is utilized to enhance efficacy of inhibitors of the PD-1/PD-L1 pathway, and/or the CD28/CTLA-4 pathway.

BACKGROUND

There is an increasing prevalence of cancer in human and its significantcontribution to mortality means there is a continuing need for newtherapies. Elimination of the cancer, a reduction in its size, thedisruption of its supporting vasculature, or reducing the number ofcancer cells circulating in the blood or lymph systems are goals ofcurrent cancer therapies. Mechanistically, therapies for cancer aredesigned to combat tumors or cells metastasizing from tumors typicallyrely on a cytotoxic activity. That activity might be a cytotoxic effectan active agent has itself or it might be an effect employed indirectlyby the active agent such as through the modulation of immune responses.

It is known that genetic and epigenetic changes occur in tissues as theytransform to take on the phenotype of a cancer cell. Different steps inthe malignant transformation process, including acquisition of themutator phenotype, which is associated with loss of tumor suppressoractivity, often results in the generation of neoantigens, which aresubject to immune recognition.

Various attempts have been made to help the immune system to fighttumors. One early approach involved a general stimulation of the immunesystem through the administration of bacteria (live or killed) to elicita general immune response which would also he directed against thetumor. Existing innate stimulators of immunity include BCG [1-10],lyophilized incubation mixture of group A Streptococcus pyogenes(OK-432) [11-26], CSF-470 [27, 28], as well as doses of chemotherapiesthat can selectively suppress Treg cells [29-39].

As far back as 1975 [40], it was known that administration ofnon-specific immune activators could induce local and in some casessystemic regression of cancer. For example, in one study, 6 patientswith intradermal metastases of malignant melanoma were treated withintralesional BCG. Four patients showed a good response with regressionof injected, and in some cases, uninjected lesions, whereas the othertwo developed metastatic visceral disease and died. Three of the sixpatients had complete regression of all lesions, and one exhibitedcomplete regression of untreated lesions. All remain free of disease.The fourth patient had complete regression of injected and of someuntreated lesions, but developed widespread dissemination and died.Three of four responders (i.e. those patients in whom treated lesionsdecreased in size by more than 50% for more than 1 month) showed adramatic increase in lymphocyte stimulation to melanoma antigens. Allresponders (four out of four) had a marked increase tophytohemagglutinin (PHA), whereas non responders had no increase inlymphocyte stimulation either to melanoma antigens or PHA. These datasuggested the other important point, which is that innate immuneactivation can lead to stimulation of antigen-specific T cell and B cellmediated immune responses. Recent approaches aimed at helping the immunesystem specifically to recognize tumor-specific antigens involveimmunization with cancer-specific antigens, typically combined with anadjuvant (a substance which is known to cause or enhance an immuneresponse) to the subject. Tumor specific antigens are well known andinclude the group of cancer testes antigens (CT antigens) or germ cellantigens that are reactivated in cancerous tissues. It is known that theusual lack of a powerful immune response to tumor associated antigens(TAAs) is due to a combination of factors. T cells have a key role inthe immune response, which is mediated through antigen recognition bythe T cell receptor (TCR), and they coordinate a balance betweenco-stimulatory and inhibitory signals known as immune checkpoints. Theseinhibitory signals function as natural suppressors of the immune systemas an important mechanism for for maintenance of self-tolerance and toprotect tissues from damage when the immune system is responding topathogenic infection. However, disregulated immune suppression reduceswhat could otherwise be a helpful response by the body to avoid thedevelopment of tumors. Cytokines, other stimulatory molecules such asCpG (stimulating dendritic cells). Toll-like receptor ligands and othermolecular adjuvants enhance the immune response. Co-stimulatoryinteractions involving T cells directly can be enhanced using agonisticantibodies to receptors including OX40, CD28, CD27 and CD137. Otherimmune system activating therapies include blocking and/or depletinginhibitory cells or molecules and include the use of antagonisticantibodies against what are known as immune checkpoints [41]. It isknown that immune cells express proteins that are immune checkpointsthat control and down-regulate the immune response. These are bestdefined in T lymphocytes and include PD-1, CTLA-4, TIM-3 and LAG3. Tumorcells express the ligands to these receptors. When T cells bind theligand to these proteins on the tumor cells, the T cell is turned offand does not attempt to attack the tumor cell. Thus, checkpoint immunesuppression is part of the complex strategy used by the tumor to evadethe patient's immune system and is responsible for resistance toimmunotherapy. Biopharmaceutical companies have successfully developedtherapeutic checkpoint inhibitors that block the receptor/ligandinteraction to promote the adaptive immune response to the tumor. Sixcheckpoint inhibitors are currently approved, pembrolizumab, nivolumab,atezolizumab, avelumab, durvalumab, and ipilimumab for a wide variety ofsolid tumors including melanoma, lung, bladder, gastric cancers andothers. T cells are central to the immune response to cancers and thereis interest in the field in using tumor infiltrating lymphocytes (TILs)in the treatment and understanding of cancer. Through their T cellreceptors (TCRs), T cells are reactive to specific antigens within atumor. Tumor cells carry genetic mutations, many of which contributedirectly or indirectly to malignancy. A mutation in an expressedsequence will typically result in a neoantigen, an antigen that is notknown to the immune system and thus recognized as foreign and able toelicit an immune response. The importance of TIL is that they areassociated with superior patient prognosis including in gastric cancer[42], breast cancer [43-46], melanoma [47], head and neck cancer [48],thus suggesting an active role of the immune system in contributing tocancer survival.

Unfortunately, despite the great advances in understanding of theimmune-cancer interaction, and development of novel first in class drugsaround this concept, many patients still do not respond toimmunotherapies, and in some cases, those that respond suffer fromrelapse. The current invention teaches means of augmenting efficacy ofimmunotherapies, specifically of checkpoint inhibitors, by removal oftumor derived and/or tumor microenvironment derived immunologicalblocking factors using extracorporeal means.

SUMMARY

Embodiments herein are directed to methods of enhancing the efficacy ofan immune checkpoint inhibitor administered to a patient suffering froma tumor comprising: identifying a patient suffering from a tumor;administering an immunological checkpoint inhibitor to said patient totreat said tumor or ameliorate the effects of said tumor;extracorporeally removing immunological blocking factors that inhibitthe effectiveness of said immunological checkpoint inhibitor in anamount sufficient to augment the efficacy of said immunologicalcheckpoint inhibitor in either treating or ameliorating the effects ofsaid tumor, wherein said extracorporeal removal is conducted at a timeselected from the group consisting of: before, concurrently, andsubsequent to the administration of said immunological checkpointinhibitor.

More specifically, disclosed herein are methods wherein said efficacy ofsaid checkpoint inhibitor is based on an endpoint selected from thegroup consisting of: a) tumor regression; b) tumor stabilization; c)reduction in tumor growth; d) inhibition of metastasis; e) stabilizationof metastasis; f) reduction of metastatic growth; g) encapsulation oftumor and/or metastasis; h) augmentation of cytokines associated withtumor inhibition; i) decrease in cytokines associated with tumorprogression; j) suppression of angiogenesis; k) augmentation of tumorinfiltrating lymphocytes; l) switch of intratumoral macrophages from M2to M1 phenotype; m) augmentation of tumor infiltrating dendritic cells;n) augmentation of tumor infiltrating killer T-cells o) reduction oftumor associated T regulatory cells; and p) reduction in tumorassociated myeloid suppressor cells.

According to further embodiments, said checkpoint inhibitor is an agentcapable of suppressing activity of a molecule selected from the groupconsisting of: PD-1, PD-L1, CTLA-4, PD-L2, LAG3, Tim3, 2B4, A2aR, ID02,B7-H3, B7-H4, BTLA, CD2, CD20, CD27, CD28, CD30, CD33, CD40, CD52, CD70,CD112, CD137, CD160, CD226, CD276, DR3, OX-40, GAL9, GITR, ICOS, HVEM,IDOL, KIR, LAIR, LIGHT, MARCO, PS, SLAM, TIGIT, VISTA, and VTCN1

According to other embodiments said immunological blocking factor issoluble TNF-alpha receptor.

According to further embodiments, disclosed herein are methods whereinsaid immunological blocking factor is selected from the group consistingof: a) soluble HLA-G; b) soluble MICA; c) interleukin-10; d)interleukin-20; e) VEGF; f) soluble IL-2 receptor; g) soluble IL-15receptor; h) interleukin-35 and i) soluble interferon gamma receptor.

According to more specific embodiments, said removal of solubleTNF-alpha receptor is performed by affinity capture to TNF-alphatrimers.

According to further embodiments said checkpoint inhibitor isadministered via a route selected from the group consisting of:intravenously, intramuscularly, parenterally, nasally, intratumorally,intraosseously, subcutaneously, sublingually, intrarectally,intrathecally, intraventricularly, orally, intraocularly, topically, orvia inhalation, nanocell and/or nanobubble injection.

According to more specific embodiments, the immunological checkpointinhibitor is selected from the group consisting of PD-1, PD-L1, andCTLA-4.

According to other embodiments, the inhibitor of PD-1 is an anti-PD-1antibody selected from the group consisting of nivolumab andpembrolizumab.

Further embodiment are directed to methods wherein the inhibitor ofPD-L1 is anti-PD-L1 antibody selected from the group consisting of:BMS-936559, durvalumab, atezolizumab, avelumab, MPDL3280A, MEDI4736,MSB0010718C, and MDX1105-01.

According to other embodiments, the inhibitor of CTLA-4 is ananti-CTLA-4 antibody selected from the group consisting of ipilimumaband tremelimumab.

According to certain embodiments, said removal of said soluble TNF-alphareceptor is performed using an extracorporeal affinity capture substratecomprising immobilized TNF-alpha molecules selected from the groupconsisting of: TNF-alpha trimers, native TNF-alpha molecules, andmutated forms of TNF-alpha, wherein said immobilized TNF-alpha moleculeson the extracorporeal affinity capture substrate have at least onebinding site capable of selectively binding to soluble TNF alphareceptor from a biological fluid.

Further methods include embodiments wherein said removal ofimmunological blocking factors is performed using an apheresis systemutilizing centrifugal plasma separation.

Additional methods include embodiments, wherein said removal ofimmunological blocking factors is performed using an apheresis systemutilizing membrane plasma separation.

Other aspects embody methods wherein enhancing efficacy of an immunecheckpoint inhibitor is accomplished by performing one or more clinicalprocedures involving the removal of tumor derived blocking factors toprepare and/or condition the patient.

Still further embodiments include methods wherein said removal ofsoluble TNF-alpha receptor is performed by affinity capture to TNF-alphatrimers.

According to more specific embodiments said checkpoint inhibitor isadministered intravenously, intramuscularly, parenterally, nasally,intratumorally, intraosseously, subcutaneously, sublingually,intrarectally, intrathecally, intraventricularly, orally, topically, orvia inhalation, nanocell and/or nanobubble injection.

According to further embodiments said extracorporeal removal of immuneblocking factors primes antigen presenting cells for enhanced ability toproduce interleukin-12 subsequent to administration of a checkpointinhibitor.

Further embodiments are directed to an immune checkpoint inhibitor foruse in a method of treating a tumor in a patient, the method comprising:identifying a patient suffering from a tumor; administering animmunological checkpoint inhibitor to said patient to treat said tumoror ameliorate the effects of said tumor; and extracorporeally removingimmunological blocking factors that inhibit the effectiveness of saidimmunological checkpoint inhibitor, wherein said extracorporeal removalis conducted at a time selected from the group consisting of: before,concurrently, and subsequent to the administration of said immunologicalcheckpoint inhibitor. All methods disclosed herein can be used with saidimmune checkpoint inhibitors.

DESCRIPTION OF THE INVENTION

The invention discloses means of augmenting therapeutic ability ofimmunological checkpoint inhibitors by removal of immunological blockingfactors through extracorporeal means. In one embodiment, the inventionrelates to the field of cancer therapy, specifically means of augmentingefficacy of cancer therapy. In particular, the invention providesmethods of generating T cell populations capable of promoting thesuppression of cancer, as well as directly killing the cancer. As usedherein, the term “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and may be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects include preventingoccurrence or recurrence of disease, alleviation of symptoms, anddiminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, lowering the rate of diseaseprogression, amelioration or palliation of the disease state, andinducing remission or improving prognosis.

The term “extracorporeal means” is defined as the use of anextracorporeal device or system through which blood or bloodconstituents obtained from a patient are passed through a device for theremoval of the immune inhibitor(s) and wherein the blood or bloodconstituents that are depleted of immune inhibitor(s) are reinfused intothe patient. The extracorporeal device is comprised of materials thatselectively binds to and captures the specified inhibitor(s) to preventthem from being reinfused into the patient.

The term “affinity capture” means the selective binding of a specificsubstance or molecule by a chemical attraction.

The term “affinity capture substrate” means a material comprising anaffinity capture molecule.

The term “antibody” includes therapeutic antibodies suitable fortreating patients; such as abagovomab, adecatumumab, afutuzumab,alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab,bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab,brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab,cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab,duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab,ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab,farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab,ganitumab, gemluzumab, girentuximab, glembatumumab, ibritumomab,igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab,iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab,lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab,mitumomab, moxetumomab, narnatumab, naptumomab, necitumumab,nimotuzumab, nofetumomabn, ocaratuzumab, ofatumumab, olaratumab,onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab,patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab,radretumab, rilotumumab, rituximab, robatumumab, satumomab,sibrotuzumab, siltuximab, simtuzumab, solitomab, tacatuzumab,taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tocilizumab,tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab,vorsetuzumab, votumumab, zalutumumab, CC49 and 3F8. In some embodimentsthe invention teaches the combination use of extracorporeal removal ofblocking factors as a means of augmenting therapeutic efficacy of thementioned antibodies. In other embodiments the combination ofantibodies, together with extracorporeal removal of blocking factors, isfurther combined with administration of checkpoint inhibitors. Theinvention is particularly of importance to therapeutic antibodies whoseactions are mediated by antibody dependent cellular toxicity. Thementioned antibodies can be utilized individually or in combination.Furthermore administration of other immune modulators is envisionedwithin the scope of the invention. Immune modulators may be activatorsof innate immunity such as toll like receptor agonists. Other immunemodulators stimulate adaptive immunity such as T and B cells.Furthermore, immune stimulation may be achieved by removal of immunesuppressive cells utilizing approaches that deplete myeloid derivedsuppressor cells, Th3 cells, T regulatory cells, type 2 neutrophils,type 2 macrophages and eosinophils.

The terms “antigen-presenting cell (s)”, “APC” or “APCs” include bothintact, whole cells as well as other molecules (all of allogeneicorigin) which are capable of inducing the presentation of one or moreantigens, preferably in association with class I MHC molecules, and alltypes of mononuclear cells which are capable of inducing an allogeneicimmune response. Preferably whole viable cells are used as APCs.Examples of suitable APCs are, but not limited to, whole cells such asmonocytes, macrophages, DCs, monocyte-derived DCs, macrophage-derivedDCs, B cells and myeloid leukemia cells e. g. cell lines THP-1, U937,HL-60 or CEM-CM3. Myeloid leukemia cells are said to provide so calledpre-monocytes. In some embodiments of the invention, tumor inducedimmaturity of antigen presenting cells is overcome by extracorporealremoval of tumor associated blocking factors. Said removal results in apredisposition of antigen presenting cells to mature in response toadministration of checkpoint inhibitors.

The terms “cancer”, “neoplasm” and “tumor” are used interchangeably andin either the singular or plural form, as appearing in the presentspecification and claims, refer to cells that have undergone a malignanttransformation that makes them pathological to the host organism.Primary cancer cells (that is, cells obtained from near the site ofmalignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e. g. by such procedures as CAT scan, magneticresonance imaging (MRI), X-ray, ultrasound or palpation. Non-limitingexamples of tumors/cancers relevant for the present invention arecarcinomas (e.g. breast cancer, prostate cancer, lung cancer, colorectalcancer, renal cancer, gastric cancer and pancreatic cancer), sarcomas(e.g. bone cancer and synovial cancer), neuro-endocrine tumors (e.g.glioblastoma, medulloblastoma and neuroblastoma), leukemias, lymphomasand squamous cell cancer (e.g. cervical cancer, vaginal cancer and oralcancer). Further, non-limiting examples of tumors/cancers relevant forthe present invention are, glioma, fibroblastoma, neurosarcoma, uterinecancer, melanoma, testicular tumors, astrocytoma, ectopichormone-producing tumor, ovarian cancer, bladder cancer, Wilm's tumor,vasoactive intestinal peptide secreting tumors, head and neck squamouscell cancer, esophageal cancer, or metastatic cancer. Prostate cancerand breast cancer are particularly preferred.

For the practice of the invention, the term “chemotherapy” is meant toencompass any non-proteinaceous (i.e, non-peptidic) chemical compounduseful in the treatment of cancer. Examples of chemotherapeutic agentsinclude reactive oxygen agents such as artimesinin and alkylating agentssuch as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates suchas busulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding alfretamine, triemylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimemylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (includingsynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (articularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cyclophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, foremustine, lomustine, nimustine,ranimustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gummaII and calicheamicin phiI1,see, e.g., Agnew, Chem. Intl. Ed. Engl, 33:183-186 (1994); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromomophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromormycins, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin(Adrarmycin™) (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as demopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogues such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replinisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;losoxantrone; fluoropyrimidine; folinic acid; podophyllinic acid;2-ethylhydrazide; procarbazine; PSK; razoxane; rhizoxin; sizofiran;spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-tricUorotriemylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethane; vindesine;dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiopeta; taxoids,e.g., paclitaxel (TAXOL®, Bristol Meyers Squibb Oncology, Princeton,N.J.) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France);chlorambucil; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitroxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; capecitabine; FOLFIRI (fluorouracil,leucovorin, and irinotecan) and pharmaceutically acceptable salts, acidsor derivatives of any of the above. In some embodiments the efficacy ofchemotherapy is augmented by utilization of extracorporeal removal ofblocking factors. Furthermore, combination of the mentionedchemotherapies may be utilized together with checkpoint inhibitors. Theinvention is particularly relevant in situations where efficacy ofchemotherapy is related to immunological activity.

The terms “extracorporeal system” and “extracorporeal removal” refer toone or methods for depleting concentrations of substances from wholeblood and/or plasma, wherein said substances are immune suppressive.Methods for depleting substances from plasma can utilize systems thatperform plasma separation using centrifugal force or separation viamembrane, including but not limited to tangential flow systems and/orcapillary means. In one embodiment, said extracorporeal means is asingle-chain TNF-alpha based affinity column, termed the “LW-02” device,which may be used in combination with the Terumo Optia apheresis system.

The term “myeloid suppressor cell” is equivalent to immature myeloidprogenitor cells, myeloid derived suppressor cells, natural suppressorcells, or immature neutrophil/monocyte precursors.

The terms “vaccine”, “immunogen”, or immunogenic composition” are usedherein to refer to a compound or composition that is capable ofconferring a degree of specific immunity when administered to a human oranimal subject. As used in this disclosure, a “cellular vaccine” or“cellular immunogen” refers to a composition comprising at least onecell population, which is optionally inactivated, as an activeingredient. The immunogens, and immunogenic compositions of thisinvention are active, which mean that they are capable of stimulating aspecific immunological response (such as an anti-tumor antigen oranti-cancer cell response) mediated at least in part by the immunesystem of the host. The immunological response may comprise antibodies,immunoreactive cells (such as helper/inducer or cytotoxic cells), or anycombination thereof, and is preferably directed towards an antigen thatis present on a tumor towards which the treatment is directed. Theresponse may be elicited or re-stimulated in a subject by administrationof either single or multiple doses. A compound or composition is“immunogenic” if it is capable of either: a) generating an immuneresponse against an antigen (such as a tumor antigen) in a naiveindividual; or b) reconstituting, boosting, or maintaining an immuneresponse in an individual beyond what would occur if the compound orcomposition was not administered. A composition is immunogenic if it iscapable of attaining either of these criteria when administered insingle or multiple doses.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, 12 amino acids or evenlonger, and in case of MHC class II peptides (e.g. elongated variants ofthe peptides of the invention) they can be as long as 15, 16, 17, 18,19, 20 or 23 or more amino acids in length. Furthermore, the term“peptide” shall include salts of a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. Preferably,the salts are pharmaceutical acceptable salts of the peptides, such as,for example, the chloride or acetate (trifluoro-acetate) salts. It hasto be noted that the salts of the peptides according to the presentinvention differ substantially from the peptides in their state(s) invivo, as the peptides are not salts in vivo. The term “peptide” shallalso include “oligopeptide”. The term “oligopeptide” is used herein todesignate a series of amino acid residues, connected one to the othertypically by peptide bonds between the alpha-amino and carbonyl groupsof the adjacent amino acids. The length of the oligopeptide is notcritical to the invention, as long as the correct epitope or epitopesare maintained therein. The oligopeptides are typically less than about30 amino acid residues in length, and greater than about 15 amino acidsin length.

The human in need thereof may be an individual who has or is suspectedof having a cancer. In some of variations, the human is at risk ofdeveloping a cancer (e.g., a human who is genetically or otherwisepredisposed to developing a cancer) and who has or has not beendiagnosed with the cancer. As used herein, an “at risk” subject is asubject who is at risk of developing cancer (e.g., a hematologicmalignancy). The subject may or may not have detectable disease, and mayor may not have displayed detectable disease prior to the treatmentmethods described herein. An at-risk subject may have one or moreso-called risk factors, which are measurable parameters that correlatewith development of cancer, such as described herein. A subject havingone or more of these risk factors has a higher probability of developingcancer than an individual without these risk factor(s). These riskfactors may include, for example, age, sex, race, diet, history ofprevious disease, presence of precursor disease, genetic (e.g.,hereditary) considerations, and environmental exposure. In someembodiments, a human at risk for cancer includes, for example, a humanwhose relatives have experienced this disease, and those whose risk isdetermined by analysis of genetic or biochemical markers. Prior historyof having cancer may also be a risk factor for instances of cancerrecurrence. In some embodiments, provided herein is a method fortreating a human who exhibits one or more symptoms associated withcancer (e.g., a hematologic malignancy). In some embodiments, the humanis at an early stage of cancer. In other embodiments, the human is at anadvanced stage of cancer.

Overall survival (OS) is defined as the time elapsed from start oftreatment until death of any cause. Progression Free Survival (PFS)(RECIST 1.1) is calculated from start of treatment until diseaseprogression or death. Objective response rate [CR (Complete Response) orPR (Partial Response) or SD (Stable Disease)] is defined as the percentof patients with best confirmed response CR or PR or SD, using CT orMRI, and determined by a central reader per RECIST 1.1. The responsemust be confirmed by a subsequent determination greater than or equal to4 weeks apart. In some instances PET is used. The evaluations andmeasurements are performed at screening, then at 8-week intervalsstarting from first treatment until PD (Progressive Disease) orinitiation of another or additional anti-tumor therapy, whichever occursfirst. In addition, scans are performed at each long-term follow-upvisit until progression.

In one embodiment of the invention patients are chosen in which a highdegree of cancer associated immune suppression present. Immunesuppression is assessed using different means known in the art and caninclude quantification of number of immune cells in circulation,quantification of activity of immune cells in circulation,quantification of the number of immune cells found intratumorally,quantification of activity of immune cells found intratumorally,quantification of the number of immune cells found peritumorally, andquantification of activity of immune cells found peritumorally. In someembodiments of the invention, quantification of immune cells comprisesidentification and assessment of activity of cells possessing tumorcytolytic and/or tumor inhibitory activity, such cells include naturalkiller cells (NK), gamma delta T cells, natural killer T cells (NKT),innate lymphoid cells, cytotoxic T lymphocytes (CTL), and helper T cells(Th). Activities of immune cells could be ability to stimulate otherimmune cells, killing activity, tumor-growth inhibitory activity, aswell as suppression of angiogenesis. Other means of assessingsuppression of immunity includes quantification of immune suppressivecells. For example, elevations in immature dendritic cells, Th2 cells,Th3 cells, myeloid suppressor cells, M2 macrophages, T regulatory cells,N2 neutrophils, and infiltration by mesenchymal stem cells possessingimmune suppressive properties, are all measurements for selectingpatients with immune suppression.

In some embodiments, extracorporeal removal of blocking factors isutilized to reduce the immune modulatory activity of myeloid suppressorcells as a means of inducing immunological activation. Myeloidsuppressor cells are believed to be similar to the “natural suppressor”cells described by the Singhal group in the 1970s. Natural suppressorcells were found to be bone marrow derived cells possessing ability toantigen-nonspecifically suppress T cell proliferation after immuneactivation [49-55], and are upregulated by cancer and pregnancy [56-63].These properties are similar to the currently described properties ofmyeloid derived suppressor cells [64].

In some embodiments of the invention, vitamin D3 is added toextracorporeal removal of blocking factors in order to augmentdifferentiation and/or loss of immune suppressive ability of saidmyeloid derived suppressor cells. Utilization of vitamin D3 to reducecancer associated immune suppression is described in this publicationand incorporated by reference [65, 66].

The invention teaches that in patients with pre-existing immunesuppression, removal of extracorporeal blocking factors may be used toincrease efficacy of checkpoint inhibitor drugs. For the practice of theinvention, various checkpoint inhibitors may be utilized together withextracorporeal removal of immunological blocking factors for enhancedtherapeutic activity. Examples of such checkpoint inhibitors include: a)Inhibitors of Programmed Death 1 (PD-1, CD279), such as nivolumab(OPDIVO®, BMS-936558, MDX1106, or MK-34775), and pembrolizumab(KEYTRUDA®, MK-3475, SCH-900475, lambrolizumab, CAS Reg. No.1374853-91-4), as well as the PD-1 blocking agents described in U.S.Pat. Nos. 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, WO03042402, WO 2008156712, WO 2010089411, WO 2010036959, WO 2011066342, WO2011159877, WO 2011082400, and WO 2011161699; b) Inhibitors ofProgrammed Death-Ligand 1 (PD-L1 also known as B7-H1 and CD274),including antibodies such as BMS-936559, MPDL3280A), MEDI14736,MSB0010718C, and MDX1105-01); also including: atezolizumab, durvalumaband avelumab; c) Inhibitors of CTLA-4, such as ipilimumab (YERVOY®,MDX-010, BMS-734016, and MDX-101), tremelimumab, antibody clone BNI3(Abcam), RNA inhibitors, including those described in WO 1999/032619, WO2001/029058, U.S. 2003/0051263, U.S. 2003/0055020, U.S. 2003/0056235,U.S. 2004/265839, U.S. 2005/0100913, U.S. 2006/0024798, U.S.2008/0050342, U.S. 2008/0081373, U.S. 2008/0248576, U.S. 2008/055443,U.S. Pat. Nos. 6,506,559, 7,282,564, 7,538,095 and 7,560,438 (eachincorporated herein by reference); d) inhibitors of PD-L2 (B7-DC,CD273), such as AMP-224 (Amplimune, Inc.) and rHIgM12B7; and e)Inhibitors of checkpoint proteins, including: LAG3, such as IMP321; TIM3(HAVCR2); 2B4; A2aR, ID02; B7H1; B7-H3 or B7H3, such as antibody MGA271;B7H4; BTLA; CD2; CD20, such as ibriturmomab tiuxetan, ofatumumab,rituximab, obinutuzumab and tositumomab; CD27, such as CDX-1127; CD28;CD30, such as brentuximab vedotin; CD33, such as gemtuzumab ozogamicin;CD40; CD52, such as alemtuzumab; CD70; CD80; CD86; CD112; CD137; CD160;CD226; CD276; DR3; OX-40 (TNFRSF.sub.4 and CD134); GAL9; GITR; such asTRX518; HAVCR2; HVEM; IDO1; ICOS (inducible T cell costimulator; CD278);such as MEDI570 (MedImmune LLC) and AMG557 (Amgen); KIR; LAIR; LIGHT;MARCO (macrophage receptor with collageneous structure); PS(phosphatidylserine); SLAM; TIGIT; VISTA; and VTCN1; or a combinationsthereof. In another variation, the checkpoint inhibitor is an inhibitorof a checkpoint protein selected from the group of PD-1, PD-L1 andCTLA-4. In another variation, the checkpoint inhibitor is selected fromthe group of an anti-PD-1 antibody, and anti-PD-L1 antibody, and ananti-CTLA-4 antibody. In one variation, the anti-PD-1 antibody isselected from the group of nivolumab, pembrolizumab, and lambrolizumab.In another variation, the anti-PD-L1 antibody is selected from the groupof as BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, and MDX1105-01. Inyet other variations, the anti-PD-L1 antibody is selected from the groupof durvalumab, atezolizumab, and avelumab. In another variation, theanti-CTLA-4 antibody is selected from the group of ipilimumab andtremelimumab. In one embodiment, the check point inhibitor is selectedfrom the group consisting of nivolumab, pembrolizumab, lambrolizumab,BMS-936559, MPDL3280A, MEDI4736, MSB0010718C, MDX1105-01, durvalumab,atezolizumab, avelumab, ipilimumab, and tremelimumab. In certainembodiment, the check point inhibitor is selected from the groupconsisting of nivolumab, pembrolizumab, lambrolizumab, durvalumab,atezolizumab, avelumab, ipilimumab, and tremelimumab. In one embodiment,the check point inhibitor is selected from the group consisting ofnivolumab, pembrolizumab, durvalumab, atezolizumab, and avelumab. Saidcheckpoint inhibitors listed may be administered via multiple methods,including but not limited intravenously, intrarrmscularly, parenterally,nasally, intratumorally, intraosseously, subcutaneously, sublingually,intrarectally, intrathecally, intraventricularly, orally, intra-ocular,topically, or via inhalation, nanocell and/or nanobubble injection. Forpractices of the invention, the enhancement of efficacy of an immunecheckpoint inhibitor may be accomplished by performing one or moreclinical procedures involving the removal of tumor derived blockingfactors to prepare and/or condition the patient. Said removal may beperformed at various time points prior to administration of saidcheckpoint inhibitor(s). The determination of time points of removal, insome embodiments, is performed based on immunological and/or oncologicalassessment of the patient. In some situations, immune activity of thepatient assessed, and used as a basis for determining the amount andfrequency of extracorporeal treatments prior to administration ofcheckpoint inhibitors. In some situations, it may be desirable tocontinue extracorporeal treatments while administering checkpointinhibitors. Furthermore, in some situations it may be desirable tocontinue extracorporeal treatment following administration of checkpointinhibitors.

In some embodiments, checkpoint inhibitor drugs are increased inefficacy by removal of extracorporeal blocking factors. Said checkpointinhibitor may be used to further increase in efficacy by addition of oneor more cancer vaccines, which is referred to as “active immunization”.In some embodiments of the invention, administration of checkpointinhibitors is performed together with active immunization. Immunizationmay take the form of peptides, proteins, altered peptide ligands, andcell therapy.

Antigens known to be found on cancer, and useful for the practice of theinvention include: epidermal growth factor receptor (EGFR, EGFR1,ErbB-1, HER1); ErbB-2 (HER2/neu), ErbB-3/HER3, ErbB-4/HER4, EGFR ligandfamily; insulin-like growth factor receptor (IGFR) family, IGF-bindingproteins (IGFBPs), IGFR ligand family (IGF-1R); platelet derived growthfactor receptor (PDGFR) family, PDGFR ligand family; fibroblast growthfactor receptor (FGFR) family, FGFR ligand family, vascular endothelialgrowth factor receptor (VEGFR) family, VEGF family; HGF receptor family;TRK receptor family; ephrin (EPH) receptor family; AXL receptor family;leukocyte tyrosine kinase (LTK) receptor family; TIE receptor family,angiopoietin 1, 2; receptor tyrosine kinase-like orphan receptor (ROR)receptor family; discoidin domain receptor (DDR) family; RET receptorfamily; KLG receptor family; RYK receptor family; MuSK receptor family;transforming growth factor alpha (TGF-alpha), TGF-alphareceptor;transforming growth factor-beta (TGF-beta), TGF-beta receptor;interleukin beta receptor alpha2 chain (IL13Ralpha2); interleukin-6(IL-6), 1L-6 receptor; interleukin-4, IL-4 receptor; cytokine receptors,Class I (hematopoietin family) and Class II (interferon/1L-10 family)receptors; tumor necrosis factor (TNF) family, TNF-alpha; tumor necrosisfactor (TNF) receptor superfamily (TNTRSF); death receptor family,TRAIL-receptor; cancer-testis (CT) antigens; lineage-specific antigens;differentiation antigens; alpha-actinin-4; ARTC1, breakpoint clusterregion-Abelson (Bcr-abl) fusion products; B-RAF; caspase-5 (CASP-5);caspase-8 (CASP-8); beta-catenin (CTNNB1); cell division cycle 27(CDC27); cyclin-dependent kinase 4 (CDK4); CDKN2A; COA-1; dek-can fusionprotein; EFTUD-2; Elongation factor 2 (ELF2); Ets variant gene 6/acutemyeloid leukemia 1 gene ETS (ETC6-AML1) fusion protein; fibronectin(FN); GPNMB; low density lipid receptor/GDP-L fucose; beta-D-galactose2-alpha-Lfucosyltraosferase (LDLR/FUT) fusion protein; HLA-A2; MLA-A11;heat shock protein 70-2 mutated (HSP70-2M); KIAA0205; MART2; melanomaubiquitous mutated 1, 2, 3 (MUM-1, 2, 3); prostatic acid phosphatase(PAP); neo-PAP; Myosin class 1; NFYC; OGT, OS-9; pml-RARalpha fusionprotein; PRDX5; PTPRK, K-ras (KRAS2); N-ras (NRAS); HRAS; RBAF600;SIRT12; SNRPD1; SYT-SSX1 or -SSX2 fusion protein; TriosephosphateIsomerase; BAGE; BAGE-1; BAGE-2, 3, 4, 5; GAGE-1, 2, 3, 4, 5, 6, 7, 8;GnT-V (aberrant N-acetyl glucosaminyl transferase V; MGAT5), HERV-K MEL,KK-LC, KM-HN-1, LAGE, LAGE-1, CTL-recognized antigen on melanoma(CAMEL), MAGE-A1 (MAGE-1); MAGE-A2; MAGE-A3; MAGE-A4; MAGE-A5; MAGE-A6;MAGE-A8; MAGE-A9; MAGE-A10; MAGE-A11; MAGE-A12; MAGE-3; MAGE-B1;MAGE-B2; MAGE-B5; MAGE-B6; MAGE-C1; MAGE-C2; mucin 1 (MUC1);MART-1/Melan-A (MLANA); gp100; gp100/Pme117 (S1LV); tyrosinase (TYR);TRP-1; HAGE; NA-88; NY-ESO-1; NY-ESO-1/LAGE-2; SAGE, Sp17; SSX-1, 2, 3,4; TRP2-1NT2; carcino-embryonic antigen (CEA); Kallikrein 4;mammaglobin-A; OA1; prostate specific antigen (PSA); prostate specificmembrane antigen; TRP-1/, 75; TRP-2 adipophilin; interferon inducibleprotein absent in melanoma 2 (AIM-2); BING-4; CPSF; cyclin D1;epithelial cell adhesion molecule (Ep-CAM); EpbA3; fibroblast growthfactor-5 (FGF-5); glycoprotein 250 (gp250intestinal carboxyl esterase(iCE); alpha-feto protein (AFP); M-CSF; mdm-2; MUCI; p53 (TP53); PBF;PRAMS; PSMA; RAGE-1; RNF43; RU2AS; SOX10; STEAP1; survivin (BIRCS);human telomerase reverse transcriptase (hTERT); telomerase; Wilms' tumorgene (WT1); SYCP1; BRDT; SPANX; XAGE; ADAM2; PAGE-5; LIP1; CTAGE-1;CSAGE; MMA1; CAGE; BORIS; HOM-TES-85; AF15q14; HCA661; LDHC; MORC;SGY-1; SPO11; TPX1; NY-SAR-35; FTHLI7; NXF2 TDRD1; TEX 15; FATE; TPTE;immunoglobulin idiotypes; Bence-Jones protein; estrogen receptors (ER);androgen receptors (AR); CD40; CD30; CD20; CD19; CD33; CD4; CD25; CD3;cancer antigen 72-4 (CA 72-4); cancer antigen 15-3 (CA 15-3); cancerantigen 27-29 (CA 27-29); cancer antigen 125 (CA 125); cancer antigen19-9 (CA 19-9); beta-human chorionic gonadotropin; 1-2 microglobulin;squamous cell carcinoma antigen; neuron-specific enolase; heat shockprotein gp96; GM2, sargramostim; CTLA-4; 707 alanine proline (707-AP);adenocarcinoma antigen recognized by T cells 4 (ART-4);carcinoembryogenic antigen peptide-1 (CAP-1); calcium-activated chloridechannel-2 (CLCA2); cyclophilin B (Cyp-B); and human signet ring tumor-2(HST-2).

In one embodiment, the invention teaches the use of removal ofextracorporeal blocking factors to increase the number of dendriticcells infiltrating tumors. The utilization of dendritic cells as animmunotherapy is known in the art and ways of using dendritic celltherapy are defined in the following examples for melanoma [67-118],soft tissue sarcoma [119], thyroid [120-122], glioma [123-144], multiplemyeloma, [145-153], lymphoma [154-156], leukemia [157-164], as well asliver [165-170], lung [171-184], ovarian [185-188], and pancreaticcancer [189-191]. In other embodiments the invention teaches the use ofextracorporeal removal of immunological blocking factors foraugmentation of existing dendritic cells to infiltrate tumors. Means ofassessing dendritic cell infiltration are known in the art and describedin the following examples: for gastric cancer [192-195], head and neckcancer [196-200], cervical cancer [201], breast cancer [202-204], lungcancer [205], colorectal cancer [206-208], liver cancer [209, 210], gallbladder cancer [211, 212], and pancreatic cancer [213].

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1-18. (canceled)
 19. A method of enhancing an efficacy of an antibodyadministered to a patient, comprising the steps of: (a) administering anantibody capable of suppressing activity of a CTLA-4 molecule; and (b)extracorporeally removing an immunological blocking factor that inhibitsthe effectiveness of the antibody from blood or a blood component of thepatient.
 20. The method of claim 19, wherein the antibody is selectedfrom the group consisting of ipilimumab, tremelimumab, and BNI3.
 21. Themethod of claim 19, wherein the immunological blocking factor is asoluble tumor necrosis factor (TNF)-alpha receptor.
 22. The method ofclaim 21, wherein step (b) is performed using an affinity capturesubstrate that comprises an immobilized TNF-alpha molecule comprisingone or more of a native TNF-alpha molecule and a mutated TNF-alphamolecule.
 23. The method of claim 22, wherein the immobilized TNF-alphamolecule is a trimer.
 24. The method of claim 19, further comprisingafter step (b) administering an antibody capable of suppressing activityof a CTLA-4 molecule one or more additional times to the patient. 25.The method of claim 19, further comprising after step (b)extracorporeally removing one or more additional times an immunologicalblocking factor that inhibits the effectiveness of the antibody fromblood or a blood component of the patient.
 26. The method of claim 19,wherein step (a) is performed before step (b).
 27. The method of claim19, wherein step (b) is performed before step (a).
 28. An extracorporealsystem for enhancing an efficacy of an antibody administered to apatient, comprising: a column configured to deplete a portion of animmunological blocking factor that reduces the efficacy of the antibodyfrom blood or a blood component of the patient; wherein the antibody iscapable of suppressing activity of a CTLA-4 molecule.
 29. The system ofclaim 28, wherein the antibody is selected from the group consisting ofipilimumab, tremelimumab, and BNI3.
 30. The system of claim 28, whereinthe immunological blocking factor is a soluble tumor necrosis factor(TNF)-alpha receptor.
 31. The system of claim 30, wherein the columncomprises an affinity capture substrate that comprises an immobilizedTNF-alpha molecule comprising one or more of a native TNF-alpha moleculeand a mutated TNF-alpha molecule.
 32. The system of claim 31, whereinthe affinity capture substrate is a trimer.